Serine O-acetytransferase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a serine O-acetyltransferase. The invention also relates to the construction of a recombinant DNA construct encoding all or a portion of the serine O-acetyltransferase, in sense or antisense orientation, wherein expression of the recombinant DNA construct results in production of altered levels of the serine O-acetyltransferase in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/292,411, filed May 21, 2001, the entire content ofwhich is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingserine O-acetyltransferase in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Sulfate assimilation is the process by which environmental sulfuris fixed into organic sulfur for use in cellular metabolism. The twomajor end products of this process are the essential amino acidscysteine and methionine. These amino acids are limiting in food andfeed; they cannot be synthesized by animals and thus must be acquiredfrom plant sources. Increasing the level of these amino acids in feedproducts is thus of major economic value. Key to that process isincreasing the level of organic sulfur available for cysteine andmethionine biosynthesis.

[0004] Multiple enzymes are involved in sulfur assimilation. Theseinclude high affinity sulfate transporter and low affinity sulfatetransporter proteins which serve to transport sulfur from the outsideenvironment across the cell membrane into the cell (Smith et al. (1995)PNAS 92(20):9373-9377). Once sulfur is in the cell, sulfateadenylyltransferase (ATP sulfurylase) (Bolchia et al. (1999) Plant Mol.Biol 39(3):527-537) catalyzes the first step in assimilation, convertingthe inorganic sulfur into an organic form, adenosine-5′ phosphosulfate(APS). Next, several enzymes further modify organic sulfur for use inthe biosynthesis of cysteine and methionine. For example,adenylylsulfate kinase (APS kinase) catalyzes the conversion of APS tothe biosynthetic intermediate PAPS (3′-phosphoadenosine-5′phosphosulfate) (Arz et al. (1994) Biochim. Biophy. Acta1218(3):447-452). APS reductase (5′ adenylyl phosphosulphate reductase)is utilized in an alternative pathway, resulting in an inorganic butcellularly bound (bound to a carrier) form of sulfur (sulfite) (Setya etal. (1996) PNAS 93(23):13383-13388). Sulfite reductase further reducesthe sulfite, still attached to the carrier, to sulfide and serineO-acetyltransferase converts serine to O-acetylserine, which will serveas the backbone to which the sulfide will be transferred to from thecarrier to form cysteine (Yonelcura-Sakakibara et al. (1998) J. Biol.Chem. 124(3):615-621 and Saito et al. (1995) J. Biol. Chem.270(27):16321-16326).

[0005] As described, each of these enzymes is involved in sulfateassimilation and the pathway leading to cysteine biosynthesis, which inturn serves as an organic sulfur donor for multiple other pathways inthe cell, including methionine biosynthesis. Together or singly theseenzymes and the genes that encode them have utility in overcoming thesulfur limitations known to exist in crop plants. It may be possible tomodulate the level of sulfur containing compounds in the cell, includingthe nutritionally critical amino acids cysteine and methionine.Specifically, their overexpression using tissue specific promoters willremove the enzyme in question as a possible limiting step, thusincreasing the potential flux through the pathway to the essential aminoacids. This will allow the engineering of plant tissues with increasedlevels of these amino acids, which now often must be added a supplementsto animal feed.

SUMMARY OF THE INVENTION

[0006] The present invention concerns isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide having serineO-acetyltransferase activity wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:3 have at least 90%sequence identity, or wherein the amino acid sequence of the polypeptideand the amino acid sequence of SEQ ID NO:5 have at least 85% sequenceidentity. It is preferred that the sequence identity to SEQ ID NO:5 beat least 90%, it is more preferred that the sequence identity to SEQ IDNO:3 or to SEQ ID NO:5 be at least 95%. The present invention alsorelates to isolated polynucleotides comprising the complement of thenucleotide sequence. More specifically, the present invention concernsisolated polynucleotides encoding the polypeptide sequence of SEQ IDNO:3 or SEQ ID NO:5 or nucleotide sequences comprising the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4.

[0007] In a first embodiment, the present invention relates to anisolated polynucleotide comprising: (a) a first nucleotide sequenceencoding a first polypeptide comprising at least 200 amino acids,wherein the amino acid sequence of the first polypeptide and the aminoacid sequence of SEQ ID NO:3 have at least 90% or 95% sequence identitybased on the ClustalV alignment method, (b) a second nucleotide sequenceencoding a second polypeptide comprising at least 250 amino acids,wherein the amino acid sequence of the second polypeptide and the aminoacid sequence of SEQ ID NO:5 have at least 85%, 90% or 95% sequenceidentity based on the ClustalV alignment method, or (c) the complementof the first or second nucleotide sequence, wherein the complement andthe first or second nucleotide sequence contain the same number ofnucleotides and are 100% complementary in a pairwise alignment. Thefirst polypeptide preferably comprises the amino acid sequence of SEQ IDNO:3, and the second polypeptide preferably comprises the amino acidsequence of SEQ ID NO:5. The first nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, and thesecond nucleotide sequence preferably comprises the nucleotide sequenceof SEQ ID NO:4. The polypeptide preferably has serineO-acetyltransferase activity.

[0008] In a second embodiment, the present invention concerns arecombinant DNA construct comprising any of the isolated polynucleotidesof the present invention operably linked to at least one regulatorysequence, and a cell, a plant, and a seed comprising the recombinant DNAconstruct.

[0009] In a third embodiment, the present invention relates to a vectorcomprising any of the isolated polynucleotides of the present invention.

[0010] In a fourth embodiment, the present invention concerns anisolated polynucleotide comprising a nucleotide sequence comprised byany of the polynucleotides of the first embodiment, wherein thenucleotide sequence contains at least 30, 40, or 60 nucleotides.

[0011] In a fifth embodiment, the present invention relates to a methodfor transforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention, and the celltransformed by this method. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0012] In a sixth embodiment, the present invention concerns a methodfor producing a transgenic plant comprising transforming a plant cellwith any of the isolated polynucleotides of the present invention andregenerating a plant from the transformed plant cell. The invention isalso directed to the transgenic plant produced by this method, and seedobtained from this transgenic plant.

[0013] In a seventh embodiment, the present invention relates to anisolated polypeptide comprising: (a) a first amino acid sequencecomprising at least 200 amino acids, wherein the first amino acidsequence and the amino acid sequence of SEQ ID NO:3 have at least 90% or95% sequence identity based on the ClustalV alignment method, or (b) asecond amino acid sequence comprising at least 250 amino acids, whereinthe second amino acid sequence and the amino acid sequence of SEQ IDNO:5 have at least 85%, 90%, or 95% sequence identity based on theClustalV alignment method. The first amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:3, and the second aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:5. The polypeptide preferably has serine O-acetyltransferaseactivity.

[0014] In an eighth embodiment, the invention concerns a method forisolating a polypeptide encoded by the polynucleotide of the presentinvention comprising isolating the polypeptide from a cell containing arecombinant DNA construct comprising the polynucleotide operably linkedto at least one regulatory sequence.

[0015] In a ninth embodiment, the present invention relates to a virus,preferably a baculovirus, comprising any of the isolated polynucleotidesof the present invention or any of the recombinant DNA constructs of thepresent invention.

[0016] In a tenth embodiment, the invention concerns a method ofselecting an isolated polynucleotide that affects the level ofexpression of a gene encoding a serine O-acetyltransferase protein oractivity in a host cell, preferably a plant cell, the method comprisingthe steps of: (a) constructing an isolated polynucleotide of the presentinvention or an isolated recombinant DNA construct of the presentinvention; (b) introducing the isolated polynucleotide or the isolatedrecombinant DNA construct into a host cell; (c) measuring the level ofserine O-acetyltransferase protein or activity in the host cellcontaining the isolated polynucleotide; and (d) comparing the level ofserine O-acetyltransferase protein or activity in the host cellcontaining the isolated polynucleotide with the level of serineO-acetyltransferase protein or activity in the host cell that does notcontain the isolated polynucleotide.

[0017] In an eleventh embodiment, the invention relates to a method ofobtaining a nucleic acid fragment encoding a substantial portion of aserine O-acetyltransferase protein, preferably a plant serineO-acetyltransferase protein comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least 30(preferably at least 40, most preferably at least 60) contiguousnucleotides derived from a nucleotide sequence of SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:4, and the complement of such nucleotide sequences;and amplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode a substantial portion of a serineO-acetyltransferase protein amino acid sequence.

[0018] In a twelfth embodiment, this invention concerns a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a serine O-acetyltransferase proteincomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

[0019] In a thirteenth embodiment, this invention relates a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the recombinant DNA construct of the present invention oran expression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the serineO-acetyltransferase polynucleotide in an amount sufficient to complementa null mutant to provide a positive selection means.

[0020] In a fourteenth embodiment, this invention concerns a method ofaltering the level of expression of a serine O-acetyltransferase proteinin a host cell comprising: (a) transforming a host cell with arecombinant DNA construct of the present invention; and (b) growing thetransformed host cell under conditions that are suitable for expressionof the recombinant DNA construct wherein expression of the recombinantDNA construct results in production of altered levels of the serineO-acetyltransferase protein in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

[0021] The invention can be more fully understood from the followingdetailed description and the accompanying drawing and Sequence Listingwhich form a part of this application.

[0022]FIG. 1 (FIGS. 1A and 1B) depicts the amino acid sequence alignmentbetween the serine O-acetyltransferase from corn (SEQ ID NO:3) encodedby the nucleotide sequences derived from corn clone cr1n.pk0085.c5 (SEQID NO:1) or corn clone p0022.cglnf80r (SEQ ID NO:2), the wheat serineO-acetyltransferase (SEQ ID NO:5) encoded by the nucleotide sequence ofwheat clone wpa1c.pk015.c12 (SEQ ID NO:4), the serineO-acetyltransferase from Allium tuberosum (NCBI GenBank Identifier (GI)No. 7384806; SEQ ID NO:8), and the serine O-acetyltransferase, SAT-52,from Arabidopsis thaliana (NCBI GI No. 2146774; SEQ ID NO:9). Aminoacids which are identical among all four sequences at a given positionin the consensus sequence are indicated with an asterisk (*). Dashes areused by the program to maximize alignment of the sequences. The aminoacid residues for each sequence are numbered to the left of each line ofsequence, and to the right of the last line of sequence. The amino acidresidues of the consensus sequence are numbered below each group ofsequences.

[0023] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Serine O-Acetyltransferase SEQID NO: Plant Clone Designation (Nucleotide) (Amino Acid) Corncr1n.pk0085.c5 1 3 Corn p0022.cglnf80r 2 3 Wheat wpa1c.pk015.c12 4 5

[0024] The amino acid sequence of SEQ ID NO:3 is encoded by nucleotidesnumber 49 to 978 of SEQ ID NO:1 and also by nucleotides number 71 to1000 of SEQ ID NO:2.

[0025] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The problem to be solved was to identify polynucleotides thatencode serine O-acetyltransferase proteins. These polynucleotides may beused in plant cells to alter sulfur assimilation and the biosynthesis ofcysteine and methionine. More specifically, the polynucleotides of theinstant invention may be used to create transgenic plants where theserine O-acetyltransferase levels are altered with respect tonon-transgenic plants which would result in plants with increased levelsof cysteine and methionine. The present invention has solved thisproblem by providing polynucleotide and deduced polypeptide sequencescorresponding to novel serine O-acetyltransferase proteins from corn(Zea mays) and wheat (Triticum aestivum).

[0027] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:4, or the complement of such sequences.

[0028] The term “isolated” refers to materials, such as nucleic acidmolecules and/or proteins, which are substantially free or otherwiseremoved from components that normally accompany or interact with thematerials in a naturally occurring environment. Isolated polynucleotidesmay be purified from a host cell in which they naturally occur.Conventional nucleic acid purification methods known to skilled artisansmay be used to obtain isolated polynucleotides. The term also embracesrecombinant polynucleotides and chemically synthesized polynucleotides.

[0029] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques. A“recombinant DNA construct” comprises any of the isolatedpolynucleotides of the present invention operably linked to at least oneregulatory sequence.

[0030] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0031] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0032] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

[0033] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence of SEQ ID NOs:1, 2 or 4, and the complement of such nucleotidesequences may be used to affect the expression and/or function of aserine O-acetyltransferase in a host cell. A method of using an isolatedpolynucleotide to affect the level of expression of a polypeptide in ahost cell (eukaryotic, such as plant or yeast, prokaryotic such asbacterial) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated recombinant DNAconstruct of the present invention; introducing the isolatedpolynucleotide or the isolated recombinant DNA construct into a hostcell; measuring the level of a polypeptide or enzyme activity in thehost cell containing the isolated polynucleotide; and comparing thelevel of a polypeptide or enzyme activity in the host cell containingthe isolated polynucleotide with the level of a polypeptide or enzymeactivity in a host cell that does not contain the isolatedpolynucleotide.

[0034] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0035] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least 70%identical, preferably at least 80% identical to the amino acid sequencesreported herein. Preferred nucleic acid fragments encode amino acidsequences that are at least 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are at least 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above identities but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

[0036] It is well understood by one skilled in the art that many levelsof sequence identity are useful in identifying related polypeptidesequences. Useful examples of percent identities are 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to100%. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the ClustalV method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0037] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises.

[0038] Amino acid and nucleotide sequences can be evaluated eithermanually by one skilled in the art, or by using computer-based sequencecomparison and identification tools that employ algorithms such as BLAST(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol215:403-410; see also the explanation of the BLAST algorithm on theworld wide web site for the National Center for BiotechnologyInformation at the National Library of Medicine of the NationalInstitutes of Health). In general, a sequence of ten or more contiguousamino acids or thirty or more contiguous nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene-specific oligonucleotide probes comprising 30or more contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12 or more nucleotidesmay be used as amplification primers in PCR in order to obtain aparticular nucleic acid fragment comprising the primers. Accordingly, a“substantial portion” of a nucleotide sequence comprises a nucleotidesequence that will afford specific identification and/or isolation of anucleic acid fragment comprising the sequence. The instant specificationteaches amino acid and nucleotide sequences encoding polypeptides thatcomprise one or more particular plant proteins. The skilled artisan,having the benefit of the sequences as reported herein, may now use allor a substantial portion of the disclosed sequences for purposes knownto those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

[0039] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0040] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0041] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, recombinant DNA constructs, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

[0042] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0043] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0044] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0045] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0046] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0047] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0048] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0049] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0050] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0051] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0052] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632). A “mitochondrial signal peptide” is an amino acidsequence which directs a precursor protein into the mitochondria (Zhangand Glaser (2002) Trends Plant Sci 7:14-21).

[0053] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism. Host organisms containingthe transformed nucleic acid fragments are referred to as “transgenic”organisms. Examples of methods of plant transformation includeAgrobacterium-mediated transformation (De Blaere et al. (1987) Meth.Enzymol. 143:277; Ishida Y. et al. (1996) Nature Biotech. 14:745-750)and particle-accelerated or “gene gun” transformation technology (Kleinet al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0054] “Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The term“transformation” as used herein refers to both stable transformation andtransient transformation.

[0055] The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be used by itself or maybe used in conjunction with a vector. If a vector is used, the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art. .

[0056] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0057] “Motifs” or “subsequences” refer to short regions of conservedsequences of nucleic acids or amino acids that comprise part of a longersequence. For example, it is expected that such conserved subsequenceswould be important for function, and could be used to identify newhomologues in plants. It is expected that some or all of the elementsmay be found in a homologue. Also, it is expected that one or two of theconserved amino acids in any given motif may differ in a true homologue.“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0058] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence encoding a serine O-acetyltransferasepolypeptide having at least 90% sequence identity, based on the ClustalValignment method, when compared to the amino acid sequence of SEQ IDNO:3, or having at least 85% sequence identity, based on the ClustalValignment method, when compared to the amino acid sequence of SEQ IDNO:5. The nucleotide sequence of the isolated polynucleotide preferablycomprises the nucleotide sequence of SEQ ID NOs:1, 2 or 4.

[0059] This invention also relates to the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity in apairwise alignment.

[0060] Nucleic acid fragments encoding at least a portion of severalserine O-acetyltransferase have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0061] For example, genes encoding other serine O-acetyltransferase,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0062] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least 30 (preferably at least 40,most preferably at least 60) contiguous nucleotides derived from anucleotide sequence of SEQ ID NOs:1, 2 or 4 and the complement of suchnucleotide sequences may be used in such methods to obtain a nucleicacid fragment encoding a substantial portion of an amino acid sequenceof a polypeptide.

[0063] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0064] In another embodiment, this invention concerns viruses and hostcells comprising either the recombinant DNA constructs of the inventionas described herein or isolated polynucleotides of the invention asdescribed herein. Examples of host cells which can be used to practicethe invention include, but are not limited to, yeast, bacteria, andplants.

[0065] As was noted above, the nucleic acid fragments of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of serineO-acetyltransferase in those cells. Serine O-acetyltransferase activitycan be determined by the methods described in Urano et al. (2000) Gene257:269-277, and in PCT International Publication Number WO 00/36127.Serine O-acetyltransferase is involved in sulfate assimilation and thepathway leading to cysteine biosynthesis, which in turn serves as anorganic sulfur donor for multiple other pathways in the cell, includingmethionine biosynthesis. This enzyme and the gene(s) that encodes theprotein have utility in overcoming the sulfur limitations known to existin crop plants. It may be possible to modulate the level of sulfurcontaining compounds in the cell, including the nutritionally criticalamino acids cysteine and methionine. Specifically, their overexpressionusing tissue specific promoters will remove the enzyme in question as apossible limiting step, thus increasing the potential flux through thepathway to the essential amino acids. This will allow the engineering ofplant tissues with increases levels of these amino acids, which nowoften must be added a supplements to animal feed.

[0066] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a recombinant DNA construct in whichthe coding region is operably linked to a promoter capable of directingexpression of a gene in the desired tissues at the desired stage ofdevelopment. The recombinant DNA construct may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant recombinant DNA construct may alsocomprise one or more introns in order to facilitate gene expression.

[0067] Plasmid vectors comprising the instant isolated polynucleotide(s)(or recombinant DNA construct(s)) may be constructed. The choice ofplasmid vector is dependent upon the method that will be used totransform host plants. The skilled artisan is well aware of the geneticelements that must be present on the plasmid vector in order tosuccessfully transform, select and propagate host cells containing therecombinant DNA construct or chimeric gene. The skilled artisan willalso recognize that different independent transformation events willresult in different levels and patterns of expression (Jones et al.(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics218:78-86), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

[0068] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the recombinant DNAconstruct(s) described above may be further supplemented by directingthe coding sequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as chloroplast transit sequences(Keegstra (1989) Cell 56:247-253), signal sequences or sequencesencoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev.Plant Phys. Plant Mol. Biol. 42:21-53), nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) or mitochondrial signalsequences (Zhang and Glaser (2002) Trends Plant Sci 7:14-21) with orwithout removing targeting sequences that are already present. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of use may be discovered in thefuture.

[0069] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a recombinant DNA construct designed forco-suppression of the instant polypeptide can be constructed by linkinga gene or gene fragment encoding that polypeptide to plant promotersequences. Alternatively, a recombinant DNA construct designed toexpress antisense RNA for all or part of the instant nucleic acidfragment can be constructed by linking the gene or gene fragment inreverse orientation to plant promoter sequences. Either theco-suppression or antisense recombinant DNA constructs could beintroduced into plants via transformation wherein expression of thecorresponding endogenous genes are reduced or eliminated.

[0070] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0071] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different recombinant DNA constructs utilizingdifferent regulatory elements known to the skilled artisan. Oncetransgenic plants are obtained by one of the methods described above, itwill be necessary to screen individual transgenics for those that mosteffectively display the desired phenotype. Accordingly, the skilledartisan will develop methods for screening large numbers oftransformants. The nature of these screens will generally be chosen onpractical grounds. For example, one can screen by looking for changes ingene expression by using antibodies specific for the protein encoded bythe gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

[0072] In another embodiment, the present invention concerns a serineO-acetyltransferase polypeptide having an amino acid sequence that is atleast 90% identical, based on the ClustalV method of alignment, to theamino acid sequence of SEQ ID NO:3, or having an amino acid sequencethat is at least 85% identical, based on the ClustalV method ofalignment, to the amino acid sequence of SEQ ID NO:5. The amino acidsequence of the serine O-acetyltransferase preferably comprises theamino acid sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0073] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a recombinant DNA construct for production of the instantpolypeptides. This recombinant DNA construct could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the encoded serine O-acetyltransferase. An example of avector for high level expression of the instant polypeptides in abacterial host is provided (Example 6).

[0074] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0075] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0076] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0077] Nucleic acid probes derived from the instant nucleic acidsequences may be used in direct fluorescence in situ hybridization(FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although currentmethods of FISH mapping favor use of large clones (several kb to severalhundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements insensitivity may allow performance of FISH mapping using shorter probes.

[0078] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0079] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0080] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0081] The disclosure of each reference set forth herein is incorporatedherein by reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0082] cDNA libraries representing mRNAs from various corn (Zea mays)and wheat (Triticum aestivum) tissues were prepared. The characteristicsof the libraries are described below. TABLE 2 cDNA Libraries from Cornand Wheat Library Tissue Clone cr1n Corn Root From 7 Day Old Seedlings*cr1n.pk0085.c5 p0022 Corn Mid Rib of the Middle ¾ of the 3rd Leaf Bladep0022.cglnf80r from Green Leaves Treated with Jasmonic Acid (1 mg/ml in0.02% Tween 20) 24 Hours Before Collection* wpa1c Wheat Pre-meioticAnther wpa1c.pk015.c12

[0083] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0084] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0085] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Ty1 transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0086] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phred/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

[0087] In some of the clones the cDNA fragment corresponds to a portionof the 3′-terminus of the gene and does not cover the entire openreading frame. In order to obtain the upstream information one of twodifferent protocols are used. The first of these methods results in theproduction of a fragment of DNA containing a portion of the desired genesequence while the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

[0088] cDNA clones encoding serine O-acetyltransferase were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLASTalgorithm on the world wide web site for the National Center forBiotechnology Information at the National Library of Medicine of theNational Institutes of Health) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

[0089] ESTs submitted for analysis are compared to the genbank databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTn algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402.) against the Du Pont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the tBLASTn algorithm. ThetBLASTn algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

Example 3 Characterization of cDNA Clones Encoding SerineO-Acetyltransferase

[0090] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs toserine O-acetyltransferases from Allium cepa (NCBI GI No. 6601494; SEQID NO:6), Citrullus lanatus (NCBI GI No. 1361979; SEQ ID NO:7) andAllium tuberosum (NCBI GenBank Identifier (GI) No. 7384806; SEQ IDNO:8). Shown in Table 3 are the BLAST results for individual ESTsequences (“EST”), the sequences of the entire cDNA inserts comprisingthe indicated cDNA clones (“FIS”), the sequences of contigs assembledfrom two or more EST, FIS or PCR sequences (“Contig”), or sequencesencoding an entire protein derived from an FIS or contig (“CGS”): TABLE3 BLAST Results for Sequences Encoding Polypeptides Homologous to SerineO-Acetyltransferase BLAST Results Clone Status NCBI GI No. pLog Scorecr1n.pk0085.c5 (FIS) CGS 1361979 122.00 p0022.cglnf80r (FIS) CGS 6601494129.00 wpa1c.pk015.c12 (FIS) CGS 7384806 131.00

[0091] PCT Publication WO 00/04167 which published Jan. 27, 2000describes the isolation and initial characterization of clonescr1n.pk0085.c5 and p0022.cglnf80r but does not disclose the sequence ofthe entire cDNA inserts contained in these clones.

[0092]FIG. 1 depicts the amino acid sequence alignment between theserine O-acetyltransferase from corn (SEQ ID NO:3) encoded by thenucleotide sequences derived from corn clone cr1n.pk0085.c5 (nucleotides49 to 978 of SEQ ID NO:1) or corn clone p0022.cglnf80r (nucleotides 71to 1000 of SEQ ID NO:2), the wheat serine O-acetyltransferase (SEQ IDNO:5) encoded by the nucleotide sequence of wheat clone wpa1c.pk015.c12(nucleotides 57 to 1007 of SEQ ID NO:4), the serine O-acetyltransferasefrom Allium tuberosum (NCBI GenBank Identifier (GI) No. 7384806; SEQ IDNO:8), and the serine O-acetyltransferase, SAT-52, from Arabidopsisthaliana (NCBI GI No. 2146774; SEQ ID NO:9). Amino acids which areidentical among all four sequences at a given position in the consensussequence are indicated with an asterisk (*).

[0093] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NO:3 and SEQ IDNO:5, compared to Allium tuberosum (NCBI GenBank Identifier (GI) No.7384806; SEQ ID NO:8), and five serine O-acetyltransferase proteins fromArabidopsis thaliana (PCT International Publication Number WO 00/36127).Also listed are the cellular locations for the different serineO-acetyltransferase proteins. The polypeptides of SEQ ID NO:3 and SEQ IDNO:5 are most similar to the Allium tuberosum cytosolic serineO-acetyltransferase and the Arabidopsis thaliana cytosolic serineO-acetyltransferase, SAT52. The cytosolic nature of the polypeptides ofSEQ ID NO:3 and SEQ ID NO:5 is further indicated by the conservation ofsequence identity at the carboxy terminus (FIG. 1; Urano et al. (2000)Gene 257:269-277). TABLE 4 Percent Identity of Amino Acid SequencesDeduced From the Nucleotide Sequences of cDNA Clones EncodingPolypeptides Homologous to Serine O-Acetyltransferase HomologousCellular % Identity to % Identity to Protein GI No. Location SEQ ID NO:3 SEQ ID NO: 5 A. tuberosum 7384806 cytosol 75.1 77.2 Arabidopsis SAT522146774 cytosol 68.1 70.2 Arabidopsis SAT3 608577 cytosol 51.6 51.3Arabidopsis SAT2 5597011 chloroplast 45.8 44.2 Arabidopsis SAT4 17225592chloroplast 47.1 46.7 Arabidopsis SAT1 1184048 mitochondria 50.6 50.8

[0094] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the ClustalV method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode serine O-acetyltransferase proteins.

Example 4 Expression of Recombinant DNA Constructs in Monocot Cells

[0095] A recombinant DNA construct comprising a cDNA encoding theinstant polypeptide in sense orientation with respect to the maize 27 kDzein promoter that is located 5′ to the cDNA fragment, and the 10 kDzein 3′ end that is located 3′ to the cDNA fragment, can be constructed.The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a recombinant DNAconstruct encoding, in the 5′ to 3′ direction, the maize 27 kD zeinpromoter, a cDNA fragment encoding the instant polypeptide, and the 10kD zein 3′ region.

[0096] The recombinant DNA construct described above can then beintroduced into corn cells by the following procedure. Immature cornembryos can be dissected from developing caryopses derived from crossesof the inbred corn lines H99 and LH132. The embryos are isolated 10 to11 days after pollination when they are 1.0 to 1.5 mm long. The embryosare then placed with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0097] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0098] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0099] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0100] Seven days after bombardment the tissue can be transferred to N6medium that contains bialophos (5 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining bialophos. After 6 weeks, areas of about 1 cm in diameter ofactively growing callus can be identified on some of the platescontaining the bialophos-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0101] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Recombinant DNA Constructs in Dicot Cells

[0102] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites NcoI (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by HindIII sites.

[0103] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0104] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptide. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0105] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0106] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0107] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0108] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/IL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0109] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0110] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Recombinant DNA Constructs in Microbial Cells

[0111] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand HindIII sites was inserted at the BamHI site of pET-3a. This createdpET-3aM with additional unique cloning sites for insertion of genes intothe expression vector. Then, the NdeI site at the position oftranslation initiation was converted to an NcoI site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0112] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0113] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

[0114] Serine O-acetyltransferase activity can be determined by themethods described in Urano et al. (2000) Gene 257:269-277, and in PCTInternational Publication Number WO 00/36127.

1 9 1 1308 DNA Zea mays 1 ccgcacaccc caccggccgg ccacataggc cccgacggcgactcgaagat gacggccggg 60 cagcttctgc gcaccgagcc atcagcccag ccccagcgggtgcgccacag caccccgccg 120 gcggcactcc aagcagacat cgtgccgtcg tacccgccgcccgagtcgga cggtgacgag 180 tcgtgggtct ggtcccagat caaggcggag gcgcggcgcgacgcggacgc ggagccggcg 240 ctggcctcct tcctctacgc gacggtgctg tcgcacgcgtccctggaccg gtccctggcc 300 ttccacctgg ccaacaagct gtgctcctcc acgctgctgtcgacgctcct ctacgacctc 360 ttcgtggcgt cgctcgcgga gcacccgtcc gtccgcgcggcggcggtggc cgacctgatc 420 gccgcgcggt cgcgggaccc ggcctgcgcg ggcttcgcgcactgcctcct caactacaag 480 gggttcctgg ccgtgcaggc gcaccgcgtg gcgcacgtgctgtgggcgca gggccggcgc 540 gcgctggcgc tggcgctcca gtcccgcgtc gccgaggtcttcgccgtgga catccacccg 600 gccgccaccg tcggcagggg catcctgctc gaccacgccacgggcgtcgt cgtcggggag 660 acggccgtcg tgggcgacaa cgtctccata ctccaccacgtgacgctggg cggcaccggc 720 aaggcggtgg gcgaccggca ccccaagatc ggggacggcgtgctcatcgg cgccggcgcg 780 accgtcctcg gaaacgtcag gatcggcgcc ggcgccaaggtcggcgccgg gtccgtcgtg 840 ctcatcgacg tgccgcccag gagcaccgcc gtggggaaccccgccaggct gatcggcggg 900 aagaagggcg aggaggtgat gccgggggag tccatggaccacacctcctt catacagcag 960 tggtcggact acatcatttg agcccgcaag ctagaaaaaaaaagagctcg tcttgctact 1020 gttgttatac tgctgttgcg ttttctgtgt atgtgcgtggatgtgttagc tgtatgctct 1080 tgttccagtg aggtgaaccg tggacatgct ggtgtggtgtccagaaagat atgctcaaag 1140 ttcgctctgt aattttcgaa gcagatgaac tgtgttactactttttactc tagtaaaaac 1200 tgtttctttg gctcaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaa 1308 2 1197 DNA Zea mays 2 ggcgctgtgc gagccacacc gcccgcacaccccaccggcc ggccacatag gccccgacgg 60 cgactcgaag atgacggccg ggcagcttctgcgcaccgag ccatcagccc agccccagcg 120 ggtgcgccac agcaccccgc cggcggcactccaagcagac atcgtgccgt cgtacccgcc 180 gcccgagtcg gacggtgacg agtcgtgggtctggtcccag atcaaggcgg aggcgcggcg 240 cgacgcggac gcggagccgg cgctggcctccttcctctac gcgacggtgc tgtcgcacgc 300 gtccctggac cggtccctgg ccttccacctggccaacaag ctgtgctcct ccacgctgct 360 gtcgacgctc ctctacgacc tcttcgtggcgtcgctcgcg gagcacccgt ccgtccgcgc 420 ggcggcggtg gccgacctga tcgccgcgcggtcgcgggac ccggcctgcg cgggcttcgc 480 gcactgcctc ctcaactaca aggggttcctggccgtgcag gcgcaccgcg tggcgcacgt 540 gctgtgggcg cagggccggc gcgcgctggcgctggcgctc cagtcccgcg tcgccgaggt 600 cttcgccgtg gacatccacc cggccgccaccgtcggcagg ggcatcctgc tcgaccacgc 660 cacgggcgtc gtcgtcgggg agacggccgtcgtgggcgac aacgtctcca tactccacca 720 cgtgacgctg ggcggcaccg gcaaggcggtgggcgaccgg caccccaaga tcggggacgg 780 cgtgctcatc ggcgccggcg cgaccgtcctcggaaacgtc aggatcggcg ccggcgccaa 840 ggtcggcgcc gggtccgtcg tgctcatcgacgtgccgccc aggagcaccg ccgtggggaa 900 ccccgccagg ctgatcggcg ggaagaagggcgaggaggtg atgccggggg agtccatgga 960 ccacacctcc ttcatacagc agtggtcggactacatcatt tgagcccgca agctagaaaa 1020 aaaaagagct cgtcttgcta ctgttgttatactgctgttg cgttttctgt gtatgtgcgt 1080 ggatgtgtta gctgtatgct cttgttccagtgaggtgaac cgtggacatg ctggtgtggt 1140 gtccagaaag atatgctcaa agttcgctctgtaattttcg aaaaaaaaaa aaaaaaa 1197 3 310 PRT Zea mays 3 Met Thr Ala GlyGln Leu Leu Arg Thr Glu Pro Ser Ala Gln Pro Gln 1 5 10 15 Arg Val ArgHis Ser Thr Pro Pro Ala Ala Leu Gln Ala Asp Ile Val 20 25 30 Pro Ser TyrPro Pro Pro Glu Ser Asp Gly Asp Glu Ser Trp Val Trp 35 40 45 Ser Gln IleLys Ala Glu Ala Arg Arg Asp Ala Asp Ala Glu Pro Ala 50 55 60 Leu Ala SerPhe Leu Tyr Ala Thr Val Leu Ser His Ala Ser Leu Asp 65 70 75 80 Arg SerLeu Ala Phe His Leu Ala Asn Lys Leu Cys Ser Ser Thr Leu 85 90 95 Leu SerThr Leu Leu Tyr Asp Leu Phe Val Ala Ser Leu Ala Glu His 100 105 110 ProSer Val Arg Ala Ala Ala Val Ala Asp Leu Ile Ala Ala Arg Ser 115 120 125Arg Asp Pro Ala Cys Ala Gly Phe Ala His Cys Leu Leu Asn Tyr Lys 130 135140 Gly Phe Leu Ala Val Gln Ala His Arg Val Ala His Val Leu Trp Ala 145150 155 160 Gln Gly Arg Arg Ala Leu Ala Leu Ala Leu Gln Ser Arg Val AlaGlu 165 170 175 Val Phe Ala Val Asp Ile His Pro Ala Ala Thr Val Gly ArgGly Ile 180 185 190 Leu Leu Asp His Ala Thr Gly Val Val Val Gly Glu ThrAla Val Val 195 200 205 Gly Asp Asn Val Ser Ile Leu His His Val Thr LeuGly Gly Thr Gly 210 215 220 Lys Ala Val Gly Asp Arg His Pro Lys Ile GlyAsp Gly Val Leu Ile 225 230 235 240 Gly Ala Gly Ala Thr Val Leu Gly AsnVal Arg Ile Gly Ala Gly Ala 245 250 255 Lys Val Gly Ala Gly Ser Val ValLeu Ile Asp Val Pro Pro Arg Ser 260 265 270 Thr Ala Val Gly Asn Pro AlaArg Leu Ile Gly Gly Lys Lys Gly Glu 275 280 285 Glu Val Met Pro Gly GluSer Met Asp His Thr Ser Phe Ile Gln Gln 290 295 300 Trp Ser Asp Tyr IleIle 305 310 4 1294 DNA Triticum aestivum 4 gcccacacac ccacccaccagcagcaatcc atccatccct cgcagctccg gcggcgatgc 60 cggcgggcca gcagccaccggcgcgcgagc ccgacggcgg cgactccaac caccaccccc 120 acccgccgcc ccccacgcccgcgctcccgt ccgaggtggt gccggcctac ccgccgccgg 180 agtcggagga cgacgagtcctgggtgtgga cgcagatcaa ggcggaggcc cggcgcgacg 240 ccgacgccga gccggcgctcgcctccttcc tctacgccac ggtgctctcc cacccctccc 300 tgccccgctc cctctccttccacctcgcca acaagctctg ctcctccacc ctcctctcca 360 cgctcctcta cgacctcttcctcgcctccc tcaccgcgca cccctccctc cgcgccgccg 420 tcgtcgccga cctcctcgccgcgcgcgccc gcgaccccgc ctgcgtcgga ttctcccact 480 gcctcctcaa ctacaagggcttcctcgcca tccaggcgca ccgcgtcgcg cacgtgctct 540 gggcgcagaa ccgccgcccgctcgcgctcg ccctccagtc ccgcgtcgcc gacgtcttcg 600 ccgtcgacat ccaccccgccgccgtcgtcg gcaaggccat cctcctcgac cacgccaccg 660 gcgtcgtcat cggggagaccgccgtcgtcg gtgacaacgt ctccatcctc caccacgtca 720 ccctgggtgg gactggcaaggcggtcggcg accgccaccc caagattggg gacggcgtgc 780 tcataggtgc cggcgccacaatcctcggca acgtcatgat tggagccggg gccaagattg 840 gggctggctc cgtggtgctgatagatgtgc cggcgcggag cacggcggtg gggaaccctg 900 ccaggctcat cggagggaggaagggcgagt ccgacaagga cgaggacatg cccggagagt 960 ccatggatca cacctccttcatacggcagt ggtccgacta caccatctga gagagccatt 1020 gtccaaggtc tattactcatcctctgtatc agtaaccgtg ttgtgctacc aaatacgtag 1080 tgattttgtt ttggtattgttcgcttgtgg atgaacatca actgtagtct aatgtcaagt 1140 gtgtatggcc aattgtttcttcagctgagc gaccatgctc ggatactgat agtggatgat 1200 tgatcaatga ataattttgtgatctacaat ggatttggtt gtattttcaa tcatttgctg 1260 gattaaaaaa aaaaaaaaaaaaaaaaaaaa aaaa 1294 5 317 PRT Triticum aestivum 5 Met Pro Ala Gly GlnGln Pro Pro Ala Arg Glu Pro Asp Gly Gly Asp 1 5 10 15 Ser Asn His HisPro His Pro Pro Pro Pro Thr Pro Ala Leu Pro Ser 20 25 30 Glu Val Val ProAla Tyr Pro Pro Pro Glu Ser Glu Asp Asp Glu Ser 35 40 45 Trp Val Trp ThrGln Ile Lys Ala Glu Ala Arg Arg Asp Ala Asp Ala 50 55 60 Glu Pro Ala LeuAla Ser Phe Leu Tyr Ala Thr Val Leu Ser His Pro 65 70 75 80 Ser Leu ProArg Ser Leu Ser Phe His Leu Ala Asn Lys Leu Cys Ser 85 90 95 Ser Thr LeuLeu Ser Thr Leu Leu Tyr Asp Leu Phe Leu Ala Ser Leu 100 105 110 Thr AlaHis Pro Ser Leu Arg Ala Ala Val Val Ala Asp Leu Leu Ala 115 120 125 AlaArg Ala Arg Asp Pro Ala Cys Val Gly Phe Ser His Cys Leu Leu 130 135 140Asn Tyr Lys Gly Phe Leu Ala Ile Gln Ala His Arg Val Ala His Val 145 150155 160 Leu Trp Ala Gln Asn Arg Arg Pro Leu Ala Leu Ala Leu Gln Ser Arg165 170 175 Val Ala Asp Val Phe Ala Val Asp Ile His Pro Ala Ala Val ValGly 180 185 190 Lys Ala Ile Leu Leu Asp His Ala Thr Gly Val Val Ile GlyGlu Thr 195 200 205 Ala Val Val Gly Asp Asn Val Ser Ile Leu His His ValThr Leu Gly 210 215 220 Gly Thr Gly Lys Ala Val Gly Asp Arg His Pro LysIle Gly Asp Gly 225 230 235 240 Val Leu Ile Gly Ala Gly Ala Thr Ile LeuGly Asn Val Met Ile Gly 245 250 255 Ala Gly Ala Lys Ile Gly Ala Gly SerVal Val Leu Ile Asp Val Pro 260 265 270 Ala Arg Ser Thr Ala Val Gly AsnPro Ala Arg Leu Ile Gly Gly Arg 275 280 285 Lys Gly Glu Ser Asp Lys AspGlu Asp Met Pro Gly Glu Ser Met Asp 290 295 300 His Thr Ser Phe Ile ArgGln Trp Ser Asp Tyr Thr Ile 305 310 315 6 289 PRT Allium cepa 6 Met ProCys Ser Thr Leu Pro Ile Pro Thr Phe Pro Pro Pro Glu Ser 1 5 10 15 GluSer Asp Glu Ser Trp Val Trp Asn Gln Ile Lys Ala Glu Ala His 20 25 30 ArgAsp Ala Glu Ser Glu Pro Ala Leu Ala Ser Tyr Leu Tyr Ser Thr 35 40 45 IleIle Ser His Pro Ser Leu Ala Arg Ser Leu Ser Phe His Leu Ala 50 55 60 AsnLys Leu Cys Ser Ser Thr Leu Leu Ser Thr Ser Leu Tyr Asp Leu 65 70 75 80Phe Leu Asn Thr Leu Ser Thr Phe Pro Thr Val Leu Ser Ala Ser Val 85 90 95Ala Asp Leu Ile Ala Ala Arg His Arg Asp Pro Ala Cys Val Gly Phe 100 105110 Ser His Cys Leu Leu Asn Phe Lys Gly Phe Leu Ala Val Gln Thr Gln 115120 125 Arg Ile Ala His Val Leu Trp Ser Gln Ser Arg Arg Pro Leu Ala Leu130 135 140 Ala Leu His Ser Arg Val Ala Asp Val Leu Ser Val Asp Ile HisPro 145 150 155 160 Ala Ala Arg Ile Gly Lys Gly Ile Leu Leu Asp His AlaThr Gly Val 165 170 175 Val Ile Gly Glu Thr Ala Val Ile Gly Asn Asn ValSer Ile Leu His 180 185 190 His Val Thr Leu Gly Gly Thr Gly Lys Ala GlyGly Asp Arg His Pro 195 200 205 Lys Ile Gly Asp Gly Val Leu Ile Gly AlaGly Ala Thr Ile Leu Gly 210 215 220 Asn Ile Arg Ile Gly Ala Gly Ala LysVal Gly Ala Gly Ser Val Val 225 230 235 240 Leu Ile Asp Val Pro Pro ArgThr Thr Ala Val Gly Asn Pro Ala Arg 245 250 255 Leu Ile Gly Gly Lys GluLys Pro Ser Val His Glu Asp Val Pro Gly 260 265 270 Glu Ser Met Asp HisThr Ser Phe Ile Ser Glu Trp Ser Asp Tyr Ile 275 280 285 Ile 7 294 PRTCitrullus lanatus 7 Met Pro Val Gly Glu Leu Arg Phe Ser Ser Gln Ser SerThr Thr Val 1 5 10 15 Val Glu Ser Thr Thr Asn Asn Asp Glu Thr Trp LeuTrp Gly Gln Ile 20 25 30 Lys Ala Glu Ala Arg Arg Asp Ala Glu Ser Glu ProAla Leu Ala Ser 35 40 45 Tyr Leu Tyr Ser Thr Ile Leu Ser His Ser Ser LeuGlu Arg Ser Leu 50 55 60 Ser Phe His Leu Gly Asn Lys Leu Cys Ser Ser ThrLeu Leu Ser Thr 65 70 75 80 Leu Leu Tyr Asp Leu Phe Leu Asn Ala Phe SerThr Asp Tyr Cys Leu 85 90 95 Arg Ser Ala Val Val Ala Asp Leu Gln Ala AlaArg Glu Arg Asp Pro 100 105 110 Ala Cys Val Ser Phe Ser His Cys Leu LeuAsn Tyr Lys Gly Phe Leu 115 120 125 Ala Cys Gln Ala His Arg Val Ala HisLys Leu Trp Asn Gln Ser Arg 130 135 140 Arg Pro Leu Ala Leu Ala Leu GlnSer Arg Ile Ala Asp Val Phe Ala 145 150 155 160 Val Asp Ile His Pro AlaAla Arg Ile Gly Lys Gly Ile Leu Phe Asp 165 170 175 His Ala Thr Gly ValVal Val Gly Glu Thr Ala Val Ile Gly Asn Asn 180 185 190 Val Ser Ile LeuHis His Val Thr Leu Gly Gly Thr Gly Lys Met Cys 195 200 205 Gly Asp ArgHis Pro Lys Ile Gly Asp Gly Val Leu Ile Gly Ala Gly 210 215 220 Ala ThrIle Leu Gly Asn Val Lys Ile Gly Glu Gly Ala Lys Ile Gly 225 230 235 240Ala Gly Ser Val Val Leu Ile Asp Val Pro Pro Arg Thr Thr Ala Val 245 250255 Gly Asn Pro Ala Arg Leu Val Gly Gly Lys Glu Lys Pro Ser Gln Leu 260265 270 Glu Asp Ile Pro Gly Glu Ser Met Asp His Thr Ser Phe Ile Ser Glu275 280 285 Trp Ser Asp Tyr Ile Ile 290 8 289 PRT Allium tuberosum 8 MetPro Cys Ser Thr Val Pro Phe Pro Thr Phe Pro Pro Pro Glu Ser 1 5 10 15Glu Ser Asp Glu Ser Trp Val Trp Asn Gln Ile Lys Ala Glu Ala Arg 20 25 30Arg Asp Ala Glu Ser Glu Pro Ala Leu Ala Ser Tyr Leu Tyr Ser Thr 35 40 45Ile Ile Ser His Pro Ser Leu Ala Arg Ser Leu Ser Phe His Leu Ala 50 55 60Asn Lys Leu Cys Ser Ser Thr Leu Leu Ser Thr Ser Leu Tyr Asp Leu 65 70 7580 Phe Leu Asn Ala Leu Ser Thr Phe Pro Thr Ile Leu Ser Ala Thr Val 85 9095 Ala Asp Leu Ile Ala Ala Arg His Arg Asp Pro Ala Cys Ile Gly Phe 100105 110 Ser His Cys Leu Leu Asn Phe Lys Gly Phe Leu Ala Val Gln Thr Gln115 120 125 Arg Ile Ala His Val Leu Trp Ser Gln Ser Arg Arg Pro Leu AlaLeu 130 135 140 Ala Leu His Ser Arg Val Ala Asp Val Leu Ser Val Asp IleHis Pro 145 150 155 160 Ala Ala Arg Ile Gly Lys Gly Ile Leu Leu Asp HisAla Thr Gly Val 165 170 175 Val Ile Gly Glu Thr Ala Val Ile Gly Asn AsnVal Ser Ile Leu His 180 185 190 His Val Thr Leu Gly Gly Thr Gly Lys AlaGly Gly Asp Arg His Pro 195 200 205 Lys Ile Gly Asp Gly Val Leu Ile GlyAla Gly Ala Thr Ile Leu Gly 210 215 220 Asn Ile Arg Ile Gly Ala Gly AlaLys Ile Gly Ala Gly Ser Val Val 225 230 235 240 Leu Ile Asp Val Pro ProArg Thr Thr Ala Val Gly Asn Pro Ala Arg 245 250 255 Leu Ile Gly Gly LysGlu Lys Pro Ser Met His Glu Asp Val Pro Gly 260 265 270 Glu Ser Met AspHis Thr Ser Phe Ile Ser Glu Trp Ser Asp Tyr Ile 275 280 285 Ile 9 312PRT Arabidopsis thaliana 9 Met Pro Pro Ala Gly Glu Leu Arg His Gln SerPro Ser Lys Glu Lys 1 5 10 15 Leu Ser Ser Val Thr Gln Ser Asp Glu AlaGlu Ala Ala Ser Ala Ala 20 25 30 Ile Ser Ala Ala Ala Ala Asp Ala Glu AlaAla Gly Leu Trp Thr Gln 35 40 45 Ile Lys Ala Glu Ala Arg Arg Asp Ala GluAla Glu Pro Ala Leu Ala 50 55 60 Ser Tyr Leu Tyr Ser Thr Ile Leu Ser HisSer Ser Leu Glu Arg Ser 65 70 75 80 Ile Ser Phe His Leu Gly Asn Lys LeuCys Ser Ser Thr Leu Leu Ser 85 90 95 Thr Leu Leu Tyr Asp Leu Phe Leu AsnThr Phe Ser Ser Asp Pro Ser 100 105 110 Leu Arg Asn Ala Thr Val Ala AspLeu Arg Ala Ala Arg Val Arg Asp 115 120 125 Pro Ala Cys Ile Ser Phe SerHis Cys Leu Leu Asn Tyr Lys Gly Phe 130 135 140 Leu Ala Ile Gln Ala HisArg Val Ser His Lys Leu Trp Thr Gln Ser 145 150 155 160 Arg Lys Pro LeuAla Leu Ala Leu His Ser Arg Ile Ser Asp Val Phe 165 170 175 Ala Val AspIle His Pro Ala Ala Lys Ile Gly Lys Gly Ile Leu Leu 180 185 190 Asp HisAla Thr Gly Val Val Val Gly Glu Thr Ala Val Ile Gly Asn 195 200 205 AsnVal Ser Ile Leu His His Val Thr Leu Gly Gly Thr Gly Lys Ala 210 215 220Cys Gly Asp Arg His Pro Lys Ile Gly Asp Gly Cys Leu Ile Gly Ala 225 230235 240 Gly Ala Thr Ile Leu Gly Asn Val Lys Ile Gly Ala Gly Ala Lys Val245 250 255 Gly Ala Gly Ser Val Val Leu Ile Asp Val Pro Cys Arg Gly ThrAla 260 265 270 Val Gly Asn Pro Ala Arg Leu Val Gly Gly Lys Glu Lys ProThr Ile 275 280 285 His Asp Glu Glu Cys Pro Gly Glu Ser Met Asp His ThrSer Phe Ile 290 295 300 Ser Glu Trp Ser Asp Tyr Ile Ile 305 310

What is claimed is:
 1. An isolated polynucleotide comprising: (a) afirst nucleotide sequence encoding a first polypeptide having serineO-acetyltransferase activity, wherein the amino acid sequence of thefirst polypeptide and the amino acid sequence of SEQ ID NO:3 have atleast 90% sequence identity based on the ClustalV alignment method, or(b) a second nucleotide sequence encoding a second polypeptide havingserine O-acetyltransferase activity, wherein the amino acid sequence ofthe second polypeptide and the amino acid sequence of SEQ ID NO:5 haveat least 85% sequence identity based on the ClustalV alignment method,or (c) the complement of the nucleotide sequence of (a) or (b).
 2. Thepolynucleotide of claim 1, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:5 have at least 90%sequence identity based on the ClustalV alignment method.
 3. Thepolynucleotide of claim 1, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:3 have at least 95%sequence identity based on the ClustalV alignment method, and whereinthe amino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:5 have at least 95% sequence identity based on theClustalV alignment method.
 4. The polynucleotide of claim 1, wherein theamino acid sequence of the first polypeptide comprises the amino acidsequence of SEQ ID NO:3, and wherein the amino acid sequence of thesecond polypeptide comprises the amino acid sequence of SEQ ID NO:5. 5.The polynucleotide of claim 1 wherein the first nucleotide sequencecomprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, andwherein the second nucleotide sequence comprises the nucleotide sequenceof SEQ ID NO:4.
 6. A vector comprising the polynucleotide of claim
 1. 7.A recombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.
 8. A method fortransforming a cell, comprising transforming a cell with thepolynucleotide of claim
 1. 9. A cell comprising the recombinant DNAconstruct of claim
 7. 10. A method for producing a plant comprisingtransforming a plant cell with the polynucleotide of claim 1 andregenerating a plant from the transformed plant cell.
 11. A plantcomprising the recombinant DNA construct of claim
 7. 12. A seedcomprising the recombinant DNA construct of claim
 7. 13. An isolatedpolypeptide having serine O-acetyltransferase activity, wherein thepolypeptide comprises: (a) a first amino acid sequence, wherein thefirst amino acid sequence and the amino acid sequence of SEQ ID NO:3have at least 90% sequence identity based on the ClustalV alignmentmethod, or (b) a second amino acid sequence, wherein the second aminoacid sequence and the amino acid sequence of SEQ ID NO:5 have at least85% sequence identity based on the ClustalV alignment method.
 14. Thepolypeptide of claim 13, wherein the second amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:5 have at least 90%sequence identity based on the ClustalV alignment method.
 15. Thepolypeptide of claim 13, wherein the first amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:3 have at least 95%sequence identity based on the ClustalV alignment method, and whereinthe second amino acid sequence of the polypeptide and the amino acidsequence of SEQ ID NO:5 have at least 95% sequence identity based on theClustalV alignment method.
 16. The polypeptide of claim 13, wherein thefirst amino acid sequence of the polypeptide comprises the amino acidsequence of SEQ ID NO:3, and wherein the second amino acid sequence ofthe polypeptide comprises the amino acid sequence of SEQ ID NO:5.
 17. Amethod for isolating a polypeptide encoded by the polynucleotide ofclaim 1 from a cell comprising a recombinant DNA construct comprisingsaid polynucleotide operably linked to at least one regulatory sequence.